Light-Dependent Expression of Protochlorophyllide Oxidoreductase Gene in the Liverwort, Marchantia paleacea var. diptera

A cDNA encoding the NADPH:protochlorophyllide oxidoreductase(EC 1.6.99.1 [EC] ) was isolated from suspension-cultured cells ofthe liverwort, Marchantia paleacea var. diptera. In contrastto the situation in most higher plants, the liverwort gene wasexpressed in a light-dependent manner. ²Present address: Department of Biological Science, Facultyof Science, Kumamoto University, Kurokami, Kumamoto, 860-8555Japan.     

Collapse of Peroxide-Scavenging Systems in Apple Flower-Buds Associated with Freezing Injury

Changes in the metabolic activities of peroxide-producing systemsand peroxide-scavenging systems after freezing and thawing inflower buds of the apple, Malus pumila Mill., were studied withspecial reference to freezing injury. In flower buds of the‘McIntosh’ apple that were frozen below lethal temperatures,the activity of NADH-Cyt c reductase (EC 1.6.99.3 [EC] ), one of theenzymes in the electron-transport chains that are related tothe peroxide-producing systems, decreased slightly, while thatof Cyt c oxidase (EC 1.9.3.1 [EC] ) hardly changed. By contrast, theactivities of glucose-6-phosphate dehydrogenase (EC 1.1.1.49 [EC] ),dehydroascorbate reductase (EC 1.8.5.1 [EC] ) and ascorbate peroxidase(EC 1.11.1.11 [EC] ), which are involved in the peroxide-scavengingsystems, decreased to very low levels. The activity of glyceraldehyde-3-phosphatedehydrogenase (EC 1.2.1.12 [EC] ) also decreased markedly. However,little change was observed in the activities of hexokinase (EC2.7.1.1 [EC] ), glucosephosphate isomerase (EC 5.3.1.9 [EC] ), glutathionereductase (EC 1.6.4.2 [EC] ) and glutathione peroxidase (EC 1.11.1.9 [EC] ).Examination of substrates involved in the peroxide-scavengingsystems revealed that the levels of glucose-6-phosphate andfructoses-phosphate decreased to approximately 10^–4 to10^–5 M and 10^–5 M, respectively, and the levelsof GSH decreased to about 10^–5 M or became barely detectable.A decrease in the levels of GSSG also occurred while levelsof ascorbate rose slightly. Similar results were observed withflower buds from ‘Starking Delicious’ and ‘Jonathan’apple trees. These results suggest that the freezing injury to apple flower-budsis closely related to the collapse of the peroxide-scavengingsystems that are coupled with the pentose phosphate cycle. Theresults also suggest that the dysfunction of these peroxide-scavengingsystems is caused by H₂O₂, which may be produced during freezingand thawing. (Received March 14, 1992; Accepted June 5, 1992)     

Effect of shade treatment on biosynthesis of catechins in tea plants

When tea plants were shaded with black lawn cloth for severaldays in the field, the accumulations of (—)-epicatechin,(—)-epicatechin-3-gallate, (—)-epigallocatechinand (—)-epigallocatechin-3-gallate decreased in newlydeveloping tea shoots. Radioactive tracer studies showed thatthe conversions of glucose-U-¹⁴C, shikimic acid-G-¹⁴C and phenylalanine-U-¹⁴Cinto (—)-epicatechin and (—)-epigallocatechin moietieswere depressed by the shade treatment for tea plants but theincorporation of trans-cinnamic acid-3-¹⁴C was not affected.The treatment was found to have no significant effect on theactivities of phospho-2-keto-3-deoxy-heptonate. aldolase (EC.4.1.2.15 [EC] ), 3-dehydroquinate synthase (EC. 4.6.1.3 [EC] ), 3-dehydroquinatedehydratase (EC. 4.2.1.10 [EC] ), shikimate dehydrogenase (EC. 1.1.1.25 [EC] )and trans-cinnamate 4-monooxygenase (EC. 1.14.13.11 [EC] ) in theshoots, whereas the activity of phenylalanine ammonia-lyase(EC. 4.3.1.5 [EC] ) clearly decreased. (Received March 17, 1980; )     

Tritium labelling of amino sugars at C-2 by alkaline epimerization in tritiated water

N-Acetyl-D-[2-³H]glucosamine was synthesized from N-acetyl-D-mannosamineby alkaline 2-epimerization in pyridine containing ³H₂O andnickelous acetate. The reaction involves reversible formationof an enol intermediate and therefore also resulted in incorporationof tritium into N-acetylmannosamine. After completed reaction,the two N-acetylhexosamines were separated from other radioactiveproducts and Morgan-Elson chromogens by chromatography on acolumn of Sephadex G-10, which was eluted with 10% ethanol,and were then separated from each other by chromatography onSephadex G-15 in 0·27 M sodium borate (pH 7·8).The location of the incorporated tritium was established bytreatment of the N-acetylhexosamines with borate under the conditionsof the Morgan-Elson reaction, which converts the sugars to Kuhn'schromogen I with concomitant loss of the C-2 hydrogen. As expected,this treatment resulted in the formation of ³H₂O, indicatingthat the tritium was located at C-2. [2-³H]Glucosamine was preparedby acid hydrolysis of the labelled N-acetylglucosamine and wasconverted to [2-³H]glucosamine 6-phosphate by incubation withhexokinase and ATP. The sugar phosphate was used as a substratefor glucosamine 6-phosphate deaminase (isomerase, EC 5.3.1.10 [EC] )in a simple ³H₂O release assay. N-acetyl[2-³H]glucosamine N-acetyl[2-³H]mannosamine [2-³H]glucosamine glucosamine 6-phosphate deaminase [2-³H]mannosamine     

Chloroplast Development in 4-Thiouridine-Cultured Radish Seedlings VI. Anabolic Pathway of 4-Thiouridine

In germinating radish seeds, [U-¹⁴C]-4-thiouridine was convertedto 4-thio-UMP, 4-thio-UDP, 4-thio-UTP, 4-thio-UDP glucose and4-thiouracil, of which 4-thiouracil accounted for 60–85%.4-Thio-UTP is incorporated into RNAs of radish seedlings [Shibataet al. (1980) FEBS Lett. 119: 85]. These same metabolites werelabeled following germination of radish seeds with [2-¹⁴C]-4-thiouracil.4-Thiouridine was hydrolyzed by the uridine nucleosidase (EC3.2.2.3 [EC] ) of radish seedlings as effectively as was uridine.The activity of uridine nucleosidase was increased by germinationwith 4-thiouridine. These results are a strong indication that4-thiouridine is converted to 4-thiouracil, then to 4-thio-UMPby uracil phosphoribosyltransferase (EC 2.4.2.9 [EC] ). The alternativeformation of 4-thio-UMP from 4-thiouridine by uridine kinase(EC 2.7.1.48 [EC] ) also was suggested. A possible mechanism whichmay cause inhibition of chloroplast biogenesis in 4-thiouridine-culturedseedlings is discussed. (Received October 12, 1981; Accepted January 14, 1982)     

Activation Energies of Reactions Catalyzed by the Nitrate Reductase from Chlorella vulgaris

The thermal dependence of two of the reactions catalyzed bythe nitrate reductase from Chlorella vulgaris was determined.The activation energies for NADH:nitrate oxidoreductase (EC1.6.6.1 [EC] ) and NADH:Cytochrome c oxidoreductase (EC 1.6.99.3 [EC] )are 42.1 kJ?mol^–1 and 21.5 kJ?mol^–1, respectively.Since the thermal dependency of the two enzymes is different,ratios of the activities will vary with temperature. The importanceof both rigorous thermal control during nitrate reductase assaysas well as the need to specify the temperature at which theratio of activities for the enzyme are clearly established. ¹Present Address: Cropping Systems Research Laboratory, USDA-ARS,Route 3, Box 215, Lubbock, TX 79401, U.S.A. (Received November 25, 1987; Accepted March 2, 1988)     

Glutamine synthesis in germinating seeds of Cucurbita moschata

During germination, an increase in glutamine and glutamine synthetase[L-glutamate: ammonia ligase (ADP), EC 6.3.1.2 [EC] ] occurred inthe cotyledons reaching a maximum at 4 to 6 days. The enzymehad a Km of 4.5 nun for L-glutamate, and 0.67 mu for hydroxylamine.Hydroxylamine exhibited substrate inhibition kinetics. The enzymewas inhibited by calcium ion, fluoride ion and p-hydroxymercuribenzoatebut not by EDTA, sodium ion or chloride ion. The sulf hydrylinhibition was reversed by dithiothreitol. In vivo synthesisof glutamine-¹⁴C from glutamate-¹⁴C was found to parallel theincrease in glutamine content and the in vitro glutamine synthetaseactivity during germination. ¹ Present address: Department of Biology, Mercyhurst College,Erie, Pennsylvania 16501, U.S.A. (Received June 12, 1971; )     

Molecular Characterisation of the 76 kDa Iron-Sulphur Protein Subunit of Potato Mitochondrial Complex I

Genes encoding subunits of complex I (EC 1.6.5.3 [EC] ) of the mitochondrialrespiratory chain vary in their locations between the mitochondrialand nuclear genomes in different organisms, whereas genes fora homologous multisub-unit complex in chloroplasts have to dateonly been found on the plastid genome. In potato (Solatium tuberosumL.), the gene coding for the mitochondrial 76 kDa iron-sulphurprotein is identified in the nuclear genome. The gene is transcribedinto polyadenylated mRNA which is most abundant in flowers,and more frequent in tubers than in leaves. The amino acid sequenceis well conserved relative to the nuclear-encoded 75 kDa and78 kDa subunits of Bos taurus and Neurospora crassa, respectively,and to the Paracoccus denitrificans homologue, most prominentlyin the region presumed to carry the iron-sulphur clusters. Polyclonalantibodies directed against the 78 kDa complex I subunit ofN. crassa recognise the 76 kDa polypeptide in potato mitochondrialcomplex I, and additionally a polypeptide of 75 kDa in solubilisedstroma thylakoids from spinach chloroplasts. The 32 amino acidresidues long presequence of the potato mitochondrial 76 kDacomplex I subunit targets the precursor polypeptide into isolatedpotato mitochondria but not into isolated chloroplasts. Theseresults suggest that chloroplast stroma thylakoids contain aprotein similar in size and antigenicity to, but geneticallydistinct from, the mitochondrial subunit. ¹ To whom correspondence should be addressed. ⁴ Present address: Max-Planck-Institut für Molekulare Genetik,Ihnestrasse 73, D-14195, Berlin, Germany. ⁵ Present address: Bioinside GmbH, Potsdamer Strasse 18A, D-14513Teltow, Germany.     

Purification and properties of a nitrite reductase from Porphyra yezoensis Ueda

Nitrite reductase was extracted from the red alga Porphyra yezoensisUeda and purified through precipitation with ammonium sulfate,column chromatographies, and polyacrylamide gel disk electrophoresis.The enzyme preparation thus obtained showed a single band ondisk electrophoresis. The absorption spectrum had three maxima at 385 nm (Soret band),580 nm (-band), and 278 nm; the ratio of absorbance of the Soretband to the -band was 4.3. The molecular weight and the numberof amino acid residues were estimated to be 63,000 and 601,respectively. The enzyme activity was optimal at around pH 7.5, and its activitywas heat labile as indicated by reduction of activity by about70% when heated at 37°C for 10 min. The enzyme used ferredoxin and methyl viologen, but not NADP⁺or NAD⁺, as the electron carriers. Moreover, reduced forms ofthe latter two showed no effect on its activity. Km values ofthis enzyme for NO₂^–, Fd, and MV were 8.1 x 10^–4M, 4.3 x 10^–8 M, and 3.7 x 10^–4 M, respectively.Almost half of its activity was lost when potassium cyanidewas added at a concentration as low as 10^–5 M, and theKi value was 1.8 x 10^–5 M. Thus, the nitrite reductaseof Porphyra must be systematically grouped in EC 1.7.7.1 [EC] . Itresembled closely that of Chlorella, except for the amountsof some amino acids. ¹ Present address: Department of Biological Sciences, Universityof Tsukuba, Sakura-Mura, Ibaraki, 300-31 Japan. ² Present address: Department of Fisheries, College of Agricultureand Veterinary Medicine, Nihon University, Shimouma, Setagaya-ku,Tokyo, 154 Japan. (Received June 10, 1975; )     

Effect of Potassium on Sucrose and Amino Acid Release from the Seed Coat of Developing Seeds of Pisum sativum

After removal of the embryo from developing seeds of Pisum sativum,the ‘empty’ ovules (seed coats without enclosedembryo) were filled with a solution (pH 5.5) containing mannitol(usually 400 mM) to which various salts were added. A solutioncontaining two isotopes ((a) [²H]-sucrose/[–¹⁴C]aminoisobutyricacid (AIB) or (b) [³H]valine/[₁₄C]asparagine mixture) was administeredto the plant via the petiole subtending the fruiting node, and[²H]solute and [¹⁴C]solute unloading from the seed coat wasmeasured, in pulse-labelling experiments of about 5 h. The presenceof 25 or 50 mM K⁺ in the ‘empty’ ovule enhancedthe release of sucrose from the seed coat particularly duringthe first hours of the experiment, but the stimulating effectof K⁺ on the release of labelled solutes derived from aminoacids was much smaller. The presence of 25 mM CaCl₂ did notaffect the release of sucrose or amino acids from the seed coat.The effect of K⁺ on sucrose and amino acid release is explainedas an inhibition of sucrose and amino acid resorption from theseed coat apoplast into seed coat cells, after unloading fromthe seed coat unloading sites. It is suggested that amino acidrelease is much less affected by K⁺ than sucrose release, becausefar less resorption of amino acids by seed coat parenchyma cellstakes place during amino acid transport into the seed coat cavity. Pisum sativum, pea, assimilate transport, assimilate unloading, seed-coat exudate, seed development, sucrose resorption, surgical treatment     

A sulfite-dependent adenosine triphosphatase of Thiobacillus thiooxidans. Its partial purification and some properties

A sulfite-dependent ATPase [EC 3.6.1.3 [EC] ] of Thiobacillus thiooxidanswas activated and solubilized by treatment with trypsin [EC3.4.4.4 [EC] ], and purified 84-fold with a 32% recovery. It requiredboth Mg²⁺ and SO₃^2– for full activity, and its optimumpH was found at 7.5–8.0. Mn²⁺, Co²⁺, and Ca²⁺ could partiallysubstitute for Mg²⁺, while SeO₃^2– and CrO₄^2– couldpartially substitute for SO₃^2–. The enzyme hydrolyzed ATP and deoxy-ATP most rapidly and otherphosphate esters were poorer substrates. The apparent Km valuefor ATP was 0.33 mM. The enzyme activity was strongly inhibitedby 0.2 mM NaN₃ and 10 mM NaF. (Received July 27, 1977; )     

Purification of NADP-Malic Enzyme and Phosphoenolpyruvate Carboxylase from Sugar Cane Leaves

A procedure is described for the purification of phosphoenolpyruvatecarboxylase (EC 4.1.1.31 [EC] ) and NADP-dependent malic enzyme (EC1.1.1.40 [EC] ) from sugar cane leaves. Each enzyme was purified tohomogeneity as judged by sodium dodecyl sulfate-polyacrylamidegel electro-phoresis, with about 30% yield. Phosphoenolpyruvatecarboxylase was purified 54-fold. A molecular weight of 400,000and a homotetrameric structure were determined for the nativeenzyme. The purified carboxylase had a specific activity of20.0 {diaeresis}mol (mg protein)_–1 min_–1, and wasactivated by glucose-6-phosphate and inhibited by L-malate.K_m values at pH 8.0 for phosphoenolpyruvate and bicarbonatewere 0.25 and O.l0 mM, respectively. NADP-malic enzyme, 356-foldpurified, exhibited a specific activity of 71.2 {diaeresis}mol(mg protein)_–1 min_–1 and was characterized as ahomotetramer with native molecular weight of 250,000. Purifiedmalic enzyme showed an absolute specificity for NADP⁺ and requireda divalent metal ion for activity. K_m values of 0.33 and 0.008mM for L-malate and NADP⁺, respectively, were determined. Thisenzyme was inhibited by several organic acids, including ketoand amino acids; while succinate and citrate increased the enzymeactivity when assayed with 10{diaeresis}M L-malate. The effectsshown by amino acids and by citrate were dependent on pH, beinghigher at pH 8.0 than at pH 7.0. (Received October 26, 1988; Accepted February 3, 1989)     

Changes in the Cytochrome c Oxidase Activity in Response to Light Regime for Photosynthesis Observed with the Cyanophyte Synechocystis PCC 6714

Changes in the activity of cytochrome c oxidase (EC 1.9.3.1 [EC] ,Cyt-oxidase) in response to growth conditions were studied withthe cyanophyte Synechocystis PCC 6714 in relation to changesin PSI abundance induced by light regime for photosynthesis.The activity was determined with the V_max of mammalian cytochromec oxidation by isolated membranes. The activity of glucose-6-phosphate(G-6-P):NADP⁺ oxidoreductase (EC 1.1.1.49 [EC] ) was also determinedsupplementarily. Cyt-oxidase activity was enhanced by glucoseadded to the medium even when cell growth maintained mainlyby oxygenic photosynthesis. G-6-P:NADP⁺ oxidoreductase was alsoactivated by glucose. The enhanced level of Cyt-oxidase washigher under PSII light, which causes high PSI abundance, thanthat under PSI light, which causes low PSI abundance. The levelwas intermediate under hetetrotrophic conditions. Although theactivity level was low in cells grown under autotrophic conditions,the level was again lower in cells grown under PSI light thanunder PSII light. The change of Cyt-oxidase activity in responseto light regime occurred in the same direction as that for thevariation of PSI abundance. Results suggest that in SynechocystisPCC 6714, the capacity of electron turnover at the two terminalcomponents of thylakoid electron transport system, Cyt-oxidaseand PSI, changes in parallel with each other in response tothe state of thylakoid electron transport system. ¹Present address: Institute of Botany, Academia Sinica, Beijing100044, China ²Present address: Department of Botany, Utkal University, Bhubaneswar,India 751004     

Some observations on the urea-degrading enzyme of the diatom Cyclotella cryptica and the role of nickel in its production

The urea-degrading enzyme of Cyclotella cryptica was testedin crude cell-free extracts for effects from chemical reagentsknown to distinguish between urease and ATP:urea amidolyase.Inhibition of the enzyme by hydroxyurea and its indifferenceto added ATP, Mg²⁺ or K⁺ avidin or biotin clearly characterizedthe enzyme as urease (EC 3.5.1.5 [EC] ). The Cyclotella urease wasunaffected by thiourea addition, as was also the growth of thediatom in the presence of this substrate analogue. Indirectevidence was obtained from growth studies of the diatom andcorresponding urease production showing that the enzyme: (i)contains Ni²⁺ tightly bound to an apoprotein; (ii) is producedconstitutively even from growth on nitrate and does not requireextracellular urea for its synthesis, although quantitativelythe activity is greatest from growth on urea. It is concludedthat Cyclotella urease is a Ni²⁺ constitutive enzyme similarin many respects to those previously reported from Phaeodactylumtricornutwn and Tetraselmis maculata.     

Suppression of Inhibitory Effects of Copper on Enzymatic Activities by Copper-Binding Substances from Athyrium yokoscense Assayed in vitro

A metal-tolerant fern, Athyrium yokoscense, is capable of growingin highly copper-contaminated soil, but cupric chloride inhibitedthe activities of some enzymes extracted from the fern. Thefunction in the detoxification of copper of two copper-bindingsubstances was investigated by examination of their effectson various enzymes assayed in vitro, i.e. acid phosphatase (orthophosphoric-monoesterphosphohydrolase [acid optimum], EC 3.1.3.2 [EC] ), glucose-6-phosphatedehydrogenase (D-glucose-6-phosphate: NADP⁺ 1-oxidoreductase,EC 1.1.1.49 [EC] ) and isocitrate dehydrogenase (threo-Ds-isocitrate:NADP⁺ oxidoreductase [decarboxylating], EC 1.1.1.42 [EC] ). The twocopper-binding substances, whose apparent molecular weightsare 9.5 kDa and 2 kDa, were previously obtained from the solublecytoplasmic fraction of the fern root. The 9.5-kDa substance,which is a cysteine-rich peptide induced as a result of exposureof the fern to copper, was found to suppress almost entirelythe inhibitory effects of the metal on the enzymes. The suppressoractivity of the peptide was nearly as effective as that of ethylenediaminetetraaceticacid. The 2-kDa substance, which is also found in fern thathas not been exposed to copper, had a more modest suppressoractivity. These results indicate that the 9.5-kDa substancemay contribute to the copper-tolerance of the fern growing incopper-contaminated soil. (Received August 26, 1988; Accepted March 17, 1989)     

Primary Structure and Properties of Glutathione Reductase from Arabidopsis thaliana

A cDNA encoding glutathione reductase (EC 1.6.4.2 [EC] ) from Arabidopsisthaliana was cloned by immunoscreening. The amino acid sequencededuced from the nucleotide sequence agrees with the N-terminalamino acid sequence of the major isozyme (GR II) purified fromleaves of A. thaliana. The predicted polypeptide comprises anN-terminal leader sequence of 74 amino acids, which has featuresof chloroplast-targeting peptides, and a mature polypeptideof 491 residues with a molecular mass of 52.7 kDa, which showshomology with glutathione reductases from other species. TheK_m for GSSG was 44 µM and that for NADPH was 5.0 µMfor GR II at 25C. The pH optimum for GR II was 7.5 to 8.0.The native molecular mass of GR II was 110 kDa, indicating thatGR II is a homodimer. GR II had an isoelectric point of 4.8.The cDNA hybridizes with a 2.1-kb poly(A)⁺RNA from leaves ofA. thaliana. Genomic Southern analysis indicates that the genecorresponding to the cDNA is likely a single-copy gene. (Received July 1, 1993; Accepted September 8, 1993)     

Biosynthetic mechanism of glycolate in Chromatium IV. Glycolate-glycine transformation

The metabolic transformation of glycolate to glycine occurringin photosynthesizing cells of Chromatium was investigated bythe radioisotopic technique and by amino acid analysis. By analyzingthe distribution of radiocarbon upon feeding [1-¹⁴C] glycolate,[2-¹⁴C] glyoxylate and [1-¹⁴C] glycine to bacterial cells, itwas demonstrated that glycolate is converted to glycinc viaglyoxylate, and both glycolate and glycine are excreted extracellularly.Although the formation of serine was barely detected by theabove two techniques in both N₂ and O₂ atmospheres, it was foundthat ¹⁴CO₂ is evolved quite markedly from both [1-¹⁴C] glycolateand [1-¹⁴C] glycine fed to the Chromatium cells. Analyticalresults of transient changes in amino acid compositions underatmospheric changes of N₂O₂ and by the addition of exogenousglycolate in N₂ confirm the notion that glycolate is convertedto glycine. Acidic amino acids (glutamic acid and aspartic acid)appear to take part in glycine formation as amino donors. Theformation of glycine from glycolate in a N₂ atmosphere suggeststhat an unknown glycolate dehydrogenation reaction may operatein the overall process. ¹ This is paper XXXVII in the series ‘Structure and Functionof Chloroplast Proteins’. Paper XXXVI is ref. (5). Theresearch was supported in part by grants from the Ministry ofEducation of Japan (No. 111912), the Toray Science Foundation(Tokyo) and the Naito Science Foundation (Tokyo). (Received July 14, 1976; )     

Lamprothamnium, a Euryhaline Charophyte: I. OSMOTIC RELATIONS AND MEMBRANE POTENTIAL AT STEADY STATE

The charophyte Lamprothamnium papulosum (Wallr.) J. Gr. is foundat salinities varying from nearly fresh water to twice thatof sea water. It can maintain its turgor constant at 302 mosmolkg^–1 (0.73 MPa) when exposed to external osmotic pressuresof 550 to 1350 mosmol kg^–1 (1.3–3.3 MPa). Turgorshows a tendency to rise slightly at lower osmotic pressure(388 mosmol kg^–1 of turgor at 150 mosmol kg^–1 externalosmolality). K⁺ and Cl^– are the main solutes in the vacuole,and are most important in controlling internal osmotic pressure.Mg²⁺, Ca²⁺, and SO^2–₄ are present in significant amountsbut their concentrations do not change with changes in externalsalinity. Na⁺ is present in lower concentration than K⁺, andplays a minor role in regulating turgor. Sucrose is presentin significant concentrations, but changes little with changesin salinity. Two enzymes involved in sucrose metabolism, sucrosephosphate synthetase (EC 2.4.1.14 [EC] ), and sucrose synthetase (EC2.4.1.13 [EC] ) are active in whole cell extracts of Lamprothamnium.As in the fresh water charophytes, Lamprothamnium membrane potentialmay be depolarized (close to E_K) or hyperpolarized, and presumablyof electrogenic origin. Both types of potential are found atall salinities tested.     

Accumulation of Hydrogen Peroxide in Chloroplasts of SO2-Fumigated Spinach Leaves

Illuminated chloroplasts isolated from SO₂-fumigated spinachleaves accumulated more H₂O₂ than those from non-fumigated ones.This H₂O₂ formation was dependent on light and was inhibitedby DCMU. It also was depressed by cytochrome c and superoxidedismutase (EC 1.15.1.1 [EC] ). The addition of sulfite to rupturedchloroplasts isolated from non-fumigated leaves caused an H₂O₂accumulation that accompanied O₂ uptake. Spinach leaves losttheir catalase (EC 1.11.1.6 [EC] ), ascorbate peroxidase and glutathionereductase (EC 1.6.4.2 [EC] ) activities at the beginning of SO₂ fumigation,when H₂O₂ was accumulated. These results suggest that the accumulationof H₂O₂ in SO₂-fumigated spinach leaves is caused by the increasein O₂^–production, the precursor for H₂O₂, with a sulfite-mediatedchain reaction at the reducing site of photosystem I, and byinactivation of the H₂O₂ scavenging system. (Received October 7, 1981; Accepted June 16, 1982)     

Changes in some Calvin Cycle Enzymes of the Tomato during Acclimation to Irradiance

The activities of three Calvin cycle enzymes, RuBPc (E.C. 4.1.1.39 [EC] ),3PGA phosphokinase (E.C. 2.7.2.3 [EC] ) and NADP-G3P dehydrogenase(E.C. 1.2.1.13 [EC] ), and the cytoplasmic enzyme PEPc (E.C. 4.1.1.31 [EC] )together with soluble protein and chlorophyll were measuredin extracts from young tomato leaves during acclimation to achange in irradiance. Leaf area and fresh weight were also measuredto show changes due to growth during treatments. Soluble proteinhad doubled on a unit leaf area basis 7 d after transfer from100 µmol quanta m^–2s^–1 PAR (low light) to400 µmol quanta m^–2s^–1 PAR (high light). Duringthis period the protein/chlorophyll ratio rose from 4•6to 10, RuBPc activity almost doubled and PEPc almost trebled.Following the reverse transfer from high to low light, solubleprotein decreased by 30% after 7 d and the protein/chlorophyllratio fell from 12 to 5•6. There was no change in RuBPcactivity 3 d after transfer from high to low light while PEPcactivity decreased by over 30%. There was no decrease in theactivity of 3PGA phosphokinase or NADP-G3P dehydrogenase 1 dafter transfer to low light, but decreases were apparent after3 d. The extracted kinase and dehydrogenase when fully activatedwere able to phosphorylate and reduce 3PGA at more than 2•5-foldits calculated rate of synthesis in the leaf. The data are discussedin relation to changes in the CO₂ exchange of the leaf. Key words: Photosynthetic acclimation, irradiance, tomato leaf, RuBP carboxylase